Analysis method

Abstract

This analysis method includes a separation step for separating an analysis target substance contained in a sample by using chromatography, and a detection step for detecting the analysis target substance separated in the separation step. A mobile phase used for the chromatography includes: one or more oxalic acid compounds selected from the group consisting of oxalic acid, (COONR4)2, and HOOCCOONR4; and one or more monovalent carboxylic acid compounds selected from the group consisting of formic acid, acetic acid, propionic acid, HCOONR4, CH3COONR4, and CH3CH2 COONR4 (R being a hydrogen atom or an alkyl group).

Description

Analysis method

[0001] The present disclosure relates to analytical methods, and more particularly to chromatographic analytical methods.

[0002] Instruments used in chromatography analysis are made of materials with high corrosion and chemical resistance. For example, metal materials such as stainless steel are generally used for the piping and filters of liquid chromatographs, and the flow cells of detectors.

[0003] However, in fields such as biochemistry and pharmaceutical science, many samples analyzed by chromatography contain substances that are easily adsorbed to metals, such as stainless steel. It is known that adsorption of such samples to metals can cause problems such as increased noise and reduced detection sensitivity. Therefore, there is a need for a method for analyzing samples containing substances that are easily adsorbed to metals with low noise and high detection sensitivity.

[0004] Patent Document 1 describes that performing liquid chromatography using a mobile phase to which oxalic acid has been added prevents CTP, CDP, and GDP from being adsorbed to metals in piping, etc., thereby reducing noise and improving detection intensity.

[0005] International Publication No. 2020 / 090887

[0006] In chromatography, poor peak shape symmetry can lead to problems such as reduced separation performance and analytical accuracy, a smaller quantitation limit, and reduced reliability of peak integration, so there is a need for the development of a technology for improving peak shape symmetry. In the analytical method described in Patent Document 1, peak tailing can be suppressed to some extent by using a mobile phase containing oxalic acid, but it is desirable to further improve the symmetry of the peak shape.

[0007] An object of the present disclosure is to provide an analytical method that can obtain a chromatogram in which the peak shape of the analyte has excellent symmetry.

[0008] In order to solve the above problems, the present inventors have conducted extensive research. As a result, they have found that a chromatogram with excellent symmetry in the peak shape of an analyte can be obtained by performing chromatography using a mobile phase containing oxalic acid or a salt thereof and a monocarboxylic acid or a salt thereof. That is, the gist of the present disclosure is as follows.

[0009] [1] A method for analyzing a sample, comprising: a separation step of separating an analyte contained in the sample by chromatography; and a detection step of detecting the analyte separated in the separation step, wherein the mobile phase used in the chromatography comprises one or more oxalic acid compounds selected from the group consisting of oxalic acid, oxalates represented by the following general formula (1), and oxalates represented by the following general formula (2), and one or more monocarboxylic acid compounds selected from the group consisting of formic acid, acetic acid, propionic acid, formates represented by the following general formula (3), acetates represented by the following general formula (4), and propionates represented by the following general formula (5), wherein when the oxalic acid compound consists solely of oxalic acid, the monocarboxylic acid compound comprises one or more selected from the group consisting of the formates, the acetates, and the propionates, and when the monocarboxylic acid compound consists solely of one or more selected from the group consisting of formic acid, acetic acid, and propionic acid, the oxalic acid compound comprises the oxalates. (COONR ４ )2 (1) HOOCCOONR ４ (2) HCOONR ４ (3) CH ３ COONR ４ (4) CH ３ CH ２ COONR ４ (5) (In the formula, each R independently represents a hydrogen atom or an alkyl group having 1 to 20 carbon atoms.) [2] The analytical method according to [1], wherein the sample contains a compound having a phosphate group, a mixture of enantiomers, or a polycarboxylic acid compound. [3] The analytical method according to [2], wherein the compound having a phosphate group is one or more selected from the group consisting of nucleic acids, nucleic acid derivatives, nucleotides, and nucleotide derivatives. [4] The analytical method according to any of [1] to [3], wherein the chromatography is liquid chromatography or supercritical fluid chromatography. [5] The analytical method according to any of [1] to [4], wherein the detection of the analyte in the detection step is performed by absorbance detection, mass spectrometry, or a combination thereof. [6] The analytical method according to [5], wherein the ionization method in the mass spectrometry is electrospray ionization or atmospheric pressure chemical ionization. [7] The analytical method according to any of [1] to [6], wherein the mobile phase satisfies either or both of the following (a) and (b): (a) The concentration of the oxalic acid compound is 0.01 mM or more and 1.0 mM or less, and (b) the concentration of the monocarboxylic acid compound is 3.5 mM or more and 20.0 mM or less. [8] A composition used in a mobile phase for chromatography, comprising one or more oxalic acid compounds selected from the group consisting of oxalic acid, oxalates represented by the following general formula (1), and oxalates represented by the following general formula (2), and one or more monocarboxylic acid compounds selected from the group consisting of formic acid, acetic acid, propionic acid, formates represented by the following general formula (3), acetates represented by the following general formula (4), and propionates represented by the following general formula (5), wherein when the oxalic acid compound consists solely of oxalic acid, the monocarboxylic acid compound comprises one or more compounds selected from the group consisting of the formates, acetates, and propionates, and when the monocarboxylic acid compound consists solely of one or more compounds selected from the group consisting of formic acid, acetic acid, and propionic acid, the oxalic acid compound comprises the oxalates. (COONR ４ )2 (1) HOOCCOONR ４ (2) HCOONR ４ (3) CH ３ COONR ４ (4) CH ３ CH ２ COONR ４ (5) (In the formula, each R independently represents a hydrogen atom or an alkyl group having 1 to 20 carbon atoms.) [9] An analytical kit comprising the mobile phase composition according to [8].

[0010] According to the present disclosure, it is possible to provide an analytical method that can obtain a chromatogram with excellent symmetry in the peak shape of an analyte. The problems and advantages of the present disclosure are not limited to those specifically described above, but also include those that will become apparent to those skilled in the art from the entire specification.

[0011] FIG. 1 is a schematic diagram of peaks for explaining elements related to calculation of a symmetry coefficient, number of theoretical plates, and peak half-width. FIG. 2 is a schematic diagram showing an example of an analytical kit according to an embodiment of the present disclosure. FIG. 3 is a liquid chromatogram in Example 1. FIG. 4 is a liquid chromatogram in Comparative Example 1. FIG. 5 is a liquid chromatogram in Comparative Example 2. FIG. 6 is a liquid chromatogram in Example 3. FIG. 7 is a liquid chromatogram in Example 4. FIG. 8 is a liquid chromatogram in Example 5. FIG. 9 is a liquid chromatogram in Example 10. FIG. 11 is a liquid chromatogram in Example 12. FIG. 13 is a liquid chromatogram in Example 14. FIG. 15 is a liquid chromatogram in Example 16. 17 is a liquid chromatogram in Example 17. 18 is a liquid chromatogram in Example 18. 19 is a liquid chromatogram in Example 19.

[0012] The present disclosure will be described below with reference to specific embodiments. However, each configuration and combination thereof in each embodiment is merely an example, and addition, omission, substitution, and other modifications of the configuration are possible as appropriate within the scope of the gist of the present disclosure. The present disclosure is not limited by the embodiments. Furthermore, each aspect disclosed in this specification can be combined with any other feature disclosed in this specification.

[0013] In the present disclosure, the range "X to Y" means "X or more and Y or less." Furthermore, when a numerical range expressed as "X to Y" or "X or more and Y or less" is described in stages (for example, in order of preference), the upper and lower limits of each numerical range can be combined in any way.

[0014] In the present disclosure, a description such as "one or more selected from the group consisting of X, Y, and Z" means any of X, Y, Z, a combination of X and Y, a combination of X and Z, a combination of Y and Z, or a combination of X, Y, and Z.

[0015] In the present disclosure, a description such as "X such as x1, x2, and x3" lists x1, x2, and x3 as examples of X, and does not mean that X is limited to x1, x2, x3, and the like.

[0016] 1. Analysis Method A first embodiment of the present disclosure is a method for analyzing a sample, comprising: a separation step of separating analytes contained in the sample by chromatography; and a detection step of detecting the analytes separated in the separation step. The mobile phase used in the chromatography comprises one or more oxalic acid compounds selected from the group consisting of oxalic acid, oxalates represented by the following general formula (1), and oxalates represented by the following general formula (2), and one or more monocarboxylic acid compounds selected from the group consisting of formic acid, acetic acid, propionic acid, formates represented by the following general formula (3), acetates represented by the following general formula (4), and propionates represented by the following general formula (5). However, when the oxalic acid compound consists solely of oxalic acid, the monocarboxylic acid compound comprises one or more compounds selected from the group consisting of the formates, acetates, and propionates; and when the monocarboxylic acid compound consists solely of one or more compounds selected from the group consisting of formic acid, acetic acid, and propionic acid, the oxalic acid compound comprises the oxalates. That is, in this embodiment, a salt is contained in either or both of the oxalic acid compound and the monocarboxylic acid compound.

[0017] (COONR ４ )2 (1) HOOCCOONR ４ (2) HCOONR ４ (3) CH ３ COONR ４ (4) CH ３ CH ２ COONR ４ (5) In the general formulas (1) to (5), each R independently represents a hydrogen atom or an alkyl group having 1 to 20 carbon atoms.

[0018] According to this embodiment, a chromatogram with excellent symmetry of the peak shape of the analyte (hereinafter, sometimes referred to as "peak symmetry") can be obtained.

[0019] 1-1. Sample The sample to be analyzed by the analytical method according to this embodiment contains substances that are the subject of analysis in various fields such as biochemistry, medicine, pharmacy, agriculture, food, and materials.

[0020] More specifically, examples of analytes contained in a sample include compounds having a phosphate group, such as nucleic acids, ribonucleic acids, nucleic acid derivatives, nucleotides, nucleotide derivatives, sugar phosphates, sugar nucleotides, and phospholipids; enantiomeric mixtures of DL-tyrosine, DL-tryptophan, and DL-phenylalanine; and polycarboxylic acid compounds, such as isocitrate, malic acid, and tartaric acid. These may form salts. Examples of nucleic acid derivatives and nucleotide derivatives include phosphorothioates, in which the phosphate group of a nucleic acid or nucleotide is converted to a thiophosphate ester group; phosphoramidates and phosphorodiamidates, in which the phosphate group of a nucleic acid or nucleotide derivative is converted to a phosphoramidate group; locked nucleic acids (LNAs), in which the oxygen atom at the 2' position of the sugar skeleton is crosslinked with the carbon atom at the 4' position by a methylene group; 2'-O-Me nucleic acids, in which the hydroxy group at the 2' position of the sugar skeleton is methoxylated; and 2'-O-MOE nucleic acids, in which the hydroxy group at the 2' position of the sugar skeleton is methoxyethylated.

[0021] Among these, compounds having a phosphate group and polycarboxylic acid compounds are substances that are recognized to be adsorbed to metal materials forming chromatograph piping and filters, as well as detector flow cells, and the like, potentially resulting in reduced peak symmetry in chromatograms. In contrast, the analytical method according to the present embodiment can obtain chromatograms with excellent peak symmetry even for these compounds. Furthermore, while conventional analytical methods for compounds having a phosphate group and polycarboxylic acid compounds have problems such as high noise relative to the desired peak intensity and low detection sensitivity, the analytical method according to the present embodiment can suppress noise and achieve high detection sensitivity. In the examples described below, analysis of adenosine triphosphate (ATP) sodium salt, a compound having a phosphate group, using the analytical method according to the present embodiment demonstrates that a chromatogram with excellent peak symmetry, low noise, and high detection sensitivity was obtained. Therefore, the analytical method according to the present embodiment can be suitably used for analyzing compounds having a phosphate group or polycarboxylic acid compounds.

[0022] 1-2. Chromatography In the separation step, chromatography is used to separate the target substance contained in the sample. The chromatography used in this embodiment is not particularly limited, and examples thereof include liquid chromatography and supercritical fluid chromatography.

[0023] Examples of liquid chromatography include, but are not limited to, normal phase chromatography, reverse phase chromatography, size exclusion chromatography, and ion exchange chromatography. In terms of excellent detectability, the liquid chromatography is preferably reverse phase chromatography, hydrophilic interaction chromatography, or ion exchange chromatography, and more preferably reverse phase chromatography or hydrophilic interaction chromatography.

[0024] Chromatography can be performed using a commercially available chromatograph, such as a liquid chromatograph or a supercritical fluid chromatograph. Column equilibration conditions, flow rate, and other conditions can be appropriately selected depending on the column size, sample volume, type of mobile phase, and the like.

[0025] 1-3. Mobile Phase The mobile phase used in chromatography contains an oxalic acid compound and a monocarboxylic acid compound. The mobile phase is a fluid that is poured into the stationary phase together with the sample in chromatography, moves along with each component in the sample, such as the substance to be analyzed, and then elutes each component in the sample from the stationary phase. Below, the components contained in the mobile phase, including the oxalic acid compound and the monocarboxylic acid compound, are described in more detail.

[0026] (Oxalic acid compound) The oxalic acid compound contained in the mobile phase is at least one selected from the group consisting of oxalic acid and oxalate salts. The oxalate salt is represented by general formula (1) or (2). That is, the oxalate salt is a compound formed by oxalate anion and NR ４ ＋ When the mobile phase contains two or more oxalic acid compounds, the combination and ratio of the oxalic acid compounds are not particularly limited.

[0027] R is a hydrogen atom or an alkyl group, and the number of carbon atoms in the alkyl group is usually 1 or more and 20 or less, preferably 1 or more and 16 or less, more preferably 1 or more and 8 or less, even more preferably 1 or more and 4 or less, and particularly preferably 2 or more and 3 or less.

[0028] Specific examples of the alkyl group include a methyl group, an ethyl group, an n-propyl group, an isopropyl group, an n-butyl group, a sec-butyl group, a tert-butyl group, an isobutyl group, an n-pentyl group, an isopentyl group, a neopentyl group, an n-hexyl group, an n-heptyl group, an n-octyl group, a 2-ethylhexyl group, an n-nonyl group, an n-decyl group, an n-undecyl group, an n-dodecyl group, an n-tridecyl group, an n-tetradecyl group, an n-pentadecyl group, an n-hexadecyl group, an n-heptadecyl group, an n-octadecyl group, an n-nonadecyl group, an n-icosyl group, and isomers thereof.

[0029] Examples of oxalic acid salts include ammonium oxalate; monoalkylammonium salts of oxalic acid such as methylammonium salt of oxalic acid, ethylammonium salt of oxalic acid, and butylammonium salt of oxalic acid; dialkylammonium salts of oxalic acid such as dimethylammonium salt of oxalic acid, diethylammonium salt of oxalic acid, ethylmethylammonium salt of oxalic acid, and dibutylammonium salt of oxalic acid; trialkylammonium salts of oxalic acid such as trimethylammonium salt of oxalic acid, triethylammonium salt of oxalic acid, and tributylammonium salt of oxalic acid; and tetraalkylammonium salts of oxalic acid such as tetramethylammonium salt of oxalic acid, tetraethylammonium salt of oxalic acid, tetrabutylammonium salt of oxalic acid, hexadecyltrimethylammonium salt of oxalic acid, and octadecyltrimethylammonium salt of oxalic acid.

[0030] The concentration of the oxalic acid compound in the mobile phase can be selected depending on the type of sample, the desired retention time, etc., and is not particularly limited. However, it is usually 0.01 mM to 50.0 mM, preferably 0.07 mM to 50.0 mM, more preferably 0.1 mM to 50.0 mM, even more preferably 0.1 mM to 20.0 mM, even more preferably 0.1 mM to 5.0 mM, particularly preferably 0.35 mM to 1.0 mM, and most preferably 0.5 mM to 1.0 mM. By setting the concentration of the oxalic acid compound within the above range, precipitation of the oxalic acid compound can be suppressed and peak symmetry in the chromatogram can be improved. Furthermore, by setting the concentration of the oxalic acid compound above the above lower limit, peaks can be detected with sufficient intensity. Furthermore, if the concentration of the oxalic acid compound is within the above range, contamination of the mass spectrometry (MS) detector is more effectively suppressed, which is advantageous in that the applicability of the mobile phase to MS detection is increased. In the present disclosure, the "concentration of an oxalic acid compound" refers to the concentration of oxalate ions.

[0031] (Monocarboxylic acid compound) The monocarboxylic acid compound contained in the mobile phase is one or more selected from the group consisting of formic acid, acetic acid, propionic acid, formate, acetate, and propionate. Formate, acetate, and propionate are represented by general formulas (3), (4), and (5), respectively. That is, formate, acetate, and propionate are formed by the reaction of formate anion, acetate anion, and propionate anion with NR ４ ＋ It is a salt of a cation represented by NR ４ ＋ The cation represented by the formula (I) has the same meaning as the cation component of oxalate. When the mobile phase contains two or more monocarboxylic acid compounds, the combination and ratio of the monocarboxylic acid compounds are not particularly limited.

[0032] When the mobile phase contains an oxalate and one or more monocarboxylic acid salts selected from the group consisting of a formate, an acetate, and a propionate, the anion component of the oxalate and the anion component of the monocarboxylic acid may be the same or different, but are preferably the same.

[0033] Examples of formate salts include ammonium formate; monoalkylammonium salts of formate such as methylammonium salt of formate, ethylammonium salt of formate, and butylammonium salt of formate; dialkylammonium salts of formate such as dimethylammonium salt of formate, diethylammonium salt of formate, ethylmethylammonium salt of formate, and dibutylammonium salt of formate; trialkylammonium salts of formate such as trimethylammonium salt of formate, triethylammonium salt of formate, and tributylammonium salt of formate; and tetraalkylammonium salts of formate such as tetramethylammonium salt of formate, tetraethylammonium salt of formate, tetrabutylammonium salt of formate, hexadecyltrimethylammonium salt of formate, and octadecyltrimethylammonium salt of formate.

[0034] Examples of acetates include ammonium acetate; monoalkylammonium salts of acetate such as methylammonium salt of acetate, ethylammonium salt of acetate, and butylammonium salt of acetate; dialkylammonium salts of acetate such as dimethylammonium salt of acetate, diethylammonium salt of acetate, ethylmethylammonium salt of acetate, and dibutylammonium salt of acetate; trialkylammonium salts of acetate such as trimethylammonium salt of acetate, triethylammonium salt of acetate, and tributylammonium salt of acetate; and tetraalkylammonium salts of acetate such as tetramethylammonium salt of acetate, tetraethylammonium salt of acetate, tetrabutylammonium salt of acetate, hexadecyltrimethylammonium salt of acetate, and octadecyltrimethylammonium salt of acetate.

[0035] Examples of propionate salts include ammonium propionate; monoalkylammonium salts of propionic acid such as methylammonium salt of propionic acid, ethylammonium salt of propionic acid, and butylammonium salt of propionic acid; dialkylammonium salts of propionic acid such as dimethylammonium salt of propionic acid, diethylammonium salt of propionic acid, ethylmethylammonium salt of propionic acid, and dibutylammonium salt of propionic acid; trialkylammonium salts of propionic acid such as trimethylammonium salt of propionic acid, triethylammonium salt of propionic acid, and tributylammonium salt of propionic acid; and tetraalkylammonium salts of propionic acid such as tetramethylammonium salt of propionic acid, tetraethylammonium salt of propionic acid, tetrabutylammonium salt of propionic acid, hexadecyltrimethylammonium salt of propionic acid, and octadecyltrimethylammonium salt of propionic acid.

[0036] The concentration of the monocarboxylic acid compound in the mobile phase is not particularly limited and can be selected depending on the type of sample, the desired retention time, the desired peak shape, etc., but is preferably 0.1 mM or more and 500.0 mM or less, more preferably 0.1 mM or more and 100.0 mM or less, even more preferably 0.1 mM or more and 50.0 mM or less, even more preferably 1.0 mM or more and 40.0 mM or less, particularly preferably 3.5 mM or more and 20.0 mM or less, and most preferably 5.0 mM or more and 10.0 mM or less. By setting the concentration of the monocarboxylic acid compound within the above range, peak symmetry in the chromatogram can be improved. In particular, by setting the concentration of the monocarboxylic acid compound below the above upper limit, peak tailing tends to be further suppressed. In this disclosure, "concentration of the monocarboxylic acid compound" means the concentration of the monocarboxylic acid ion (when multiple types of monocarboxylic acid compounds are contained in the mobile phase, the total concentration of the monocarboxylic acid ion). In this embodiment, it is more preferable that the concentration of the oxalic acid compound is within the above-mentioned range, and the concentration of the monocarboxylic acid compound is within the above-mentioned range.

[0037] In this embodiment, the ratio of the concentration of the monocarboxylic acid compound to the concentration of the oxalic acid compound (monocarboxylic acid compound concentration (mM) / oxalic acid compound concentration (mM)) is not particularly limited, but is preferably 1 to 200, more preferably 1 to 100, even more preferably 1 to 50, even more preferably 1 to 40, particularly preferably 1 to 20, especially preferably 5 to 20, and most preferably 10 to 20. By setting the concentration ratio of the monocarboxylic acid compound to the oxalic acid compound within the above range, precipitation of the oxalic acid compound can be suppressed. Furthermore, if the concentration ratio of the monocarboxylic acid compound to the oxalic acid compound is within the above range, contamination of the MS detector is more effectively suppressed, which is effective in increasing the applicability of the mobile phase to MS detection. In this embodiment, it is more preferable that the concentration of the oxalic acid compound and the concentration of the monocarboxylic acid compound are both within the above range, and the ratio of the concentration of the monocarboxylic acid compound to the concentration of the oxalic acid compound is within the above range.

[0038] (Solvent) The mobile phase in this embodiment is a solution in which at least an oxalic acid compound and a monocarboxylic acid compound are dissolved in a solvent.

[0039] The solvent of the mobile phase when performing separation by liquid chromatography is not particularly limited, but is preferably an organic solvent that can dissolve the substance to be separated. Examples of the solvent include organic solvents, water, and mixed solvents of water and organic solvents, and preferably water or mixed solvents of water and organic solvents.

[0040] The organic solvent is not particularly limited, but examples thereof include acetonitrile, methanol, ethanol, n-propanol, 2-propanol, tetrahydrofuran, 2-methyltetrahydrofuran, dimethyl sulfoxide (DMSO), dimethylformamide (DMF), dimethylacetamide (DMAc), acetone, methyl ethyl ketone, diethyl ketone, methyl tert-butyl ether, chloroform, and dichloromethane. The organic solvent may also be a mixture of an alcohol and a hydrocarbon having 5 to 8 carbon atoms, which is commonly used in chromatography. One type of organic solvent may be used alone, or two or more types may be used in any combination and ratio.

[0041] Among these, the organic solvent is preferably one or more selected from acetonitrile, methanol, ethanol, n-propanol, 2-propanol, and tetrahydrofuran, more preferably one or more selected from methanol and acetonitrile, and even more preferably acetonitrile, in that it has high chemical stability, low viscosity, and allows the absorbance detection of the analyte.

[0042] When a mixed solvent of water and an organic solvent is used as the solvent, the mixing ratio of water and the organic solvent is not particularly limited and may be appropriately selected depending on the substance to be analyzed. Specific examples of the water content in the mixed solvent include 5% by volume or more and 95% by volume or less, 10% by volume or more and 90% by volume or less, 20% by volume or more and 80% by volume or less, 30% by volume or more and 70% by volume or less, 40% by volume or more and 60% by volume or less, and 40% by volume or more and 60% by volume or less.

[0043] Examples of the solvent for the mobile phase when performing separation by supercritical fluid chromatography include supercritical carbon dioxide, a mixture of supercritical carbon dioxide and an organic solvent, and a mixture of supercritical carbon dioxide, an organic solvent, and water. As the organic solvent contained in the mixture, those organic solvents described above as being used in the mobile phase for liquid chromatography can be suitably used.

[0044] (Optional Components) In addition to the oxalic acid compound, the monocarboxylic acid compound, and the solvent, the mobile phase in this embodiment may contain optional components to the extent that separation and detection are not hindered. Examples of optional components include pH adjusting reagents and internal standards. When the detection method in the detection step is mass spectrometry, the optional components are preferably volatile components or components that are thermally decomposed into volatile substances in the MS detector. The optional components may be used alone, or two or more may be used in any combination and ratio.

[0045] (Preparation of mobile phase) The mobile phase can be prepared by mixing an oxalic acid compound, a monocarboxylic acid compound, and a solvent. Alternatively, the mobile phase can be prepared by mixing oxalic acid, one or more monocarboxylic acids selected from the group consisting of formic acid, acetic acid, and propionic acid, and NR ４ ＋ It can also be prepared by mixing a source of a cation represented by NR with a solvent. ４ ＋ For example, a solution containing a cation source such as aqueous ammonia and a tetrabutylammonium hydroxide solution can be used to mix the cation source represented by the formula (I). When preparing a mobile phase containing the optional component, the optional component may be mixed with the above components during mixing.

[0046] The mobile phase can also be prepared by mixing a mobile phase composition described below with a solvent.

[0047] 1-4. Separation Column The separation column used in chromatography is not particularly limited as long as it is a column packed with a stationary phase, and can be appropriately selected depending on the type of sample. When an enantiomeric mixture is separated into individual enantiomers in the separation step and then detected in the subsequent detection step, a column packed with a chiral stationary phase is used as the separation column.

[0048] The stationary phase is not particularly limited, and any stationary phase such as a known stationary phase or a stationary phase equivalent thereto can be used.

[0049] Known stationary phases include a stationary phase in which a ligand having a crown ether-like cyclic structure is supported on a carrier; a stationary phase in which a polysaccharide derivative such as a cellulose derivative or an amylose derivative is supported on a carrier; a stationary phase in which an alkylsilyl group having 1 to 32 carbon atoms is chemically bonded to silica gel, such as octadecylsilyl (ODS) silica gel; or a polar phase in which a polar group such as an amide bond is embedded in the alkyl chain of the alkylsilyl group. Stationary phases called "embedded" phases; stationary phases in which a group containing a benzene ring (e.g., a phenylethyl group and a phenylhexyl group), a halogen-containing group (e.g., a pentafluorophenylpropyl, a pentachlorophenylpropyl, a pentabromophenylpropyl, etc.), a group having a cyano group (e.g., a phenylpropyl group), a group having an amide bond (e.g., an amidopropyl group), or a group having an alcoholic hydroxyl group is bonded to silica gel; stationary phases in which an aromatic polyester (e.g., polybutylene terephthalate) is supported on a carrier; stationary phases in which a heteroaromatic ring polymer (e.g., poly(4-vinylpyridine), poly(2-vinylpyridine), poly(3-vinylpyridine), poly(1-vinylimidazole), poly(vinylcarbazole), and poly(vinylpyrazine)) is supported on a carrier; stationary phases in which a hydrophilic polymer (e.g., poly(2-methacryloyloxyethylene) and stationary phases in which a carrier supports a compound selected from the group consisting of poly(2-(N-3-sulfopropyl-N,N-dimethylammonium)ethyl methacrylate), poly(3-methacryloylamino)propyltrimethylammonium chloride), poly(polyethylene glycol monomethyl ether methacrylate), poly(potassium 3-methacryloyloxypropanesulfonate), poly(2-hydroxyethyl methacrylate), poly(acrylamide), and poly(5-methacrylamide-1H-tetrazole)); stationary phases in which a carrier supports an amino acid, a cyclic oligosaccharide (e.g., cyclodextrin, cyclofructan, etc.), a cyclic oligosaccharide with a modified hydroxy group, or a macrocyclic amide (e.g., vancomycin, etc.); and stationary phases in which a carrier supports a compound having a zwitterionic functional group such as a sulfobetaine group or a phosphobetaine group.

[0050] Examples of the polysaccharide derivatives include polysaccharide phenylcarbamate derivatives in which some or all of the hydroxy groups of a polysaccharide have been modified with substituted or unsubstituted phenyl isocyanate; and polysaccharide benzoate derivatives in which some or all of the hydroxy groups of a polysaccharide have been modified with substituted or unsubstituted benzoic acid halides, etc. The modifying agent for polysaccharides is not limited to the above compounds.

[0051] In the present disclosure, "support" may mean either chemical support in which the substance to be supported is chemically bonded to the carrier, physical support in which the substance to be supported is coated or physically adsorbed onto the carrier, or a combination thereof.

[0052] The carrier is not particularly limited, and examples thereof include porous organic carriers, porous inorganic carriers, porous organic-inorganic composite carriers, surface-porous organic carriers, surface-porous inorganic carriers, and surface-porous organic-inorganic hybrid carriers. In the present disclosure, the term "porous carrier" refers to a carrier in which pores are formed throughout the entire carrier, and the term "surface-porous carrier" refers to a core-shell carrier having a structure in which a non-porous core is covered with a porous layer.

[0053] Examples of porous organic carriers include polystyrene, poly(meth)acrylamide, and poly(meth)acrylic acid ester.

[0054] Examples of porous inorganic supports include silica, alumina, magnesia, glass, kaolin, titanium oxide, zirconium oxide, silicates, and hydroxyapatite, with silica gel being preferred.

[0055] Examples of porous organic-inorganic composite carriers include organic-inorganic composite carriers formed by a sol-gel reaction between an alkoxysilane and an alkyl- or alexylene-substituted alkoxysilane compound.

[0056] 1-5. Detection Method In this embodiment, the detection step involves detecting the analyte separated in the separation step. The detection method for the analyte is not particularly limited and can be appropriately selected depending on the type of analyte, the purpose of the analysis, and the like. For example, detection methods include absorbance detection such as ultraviolet absorbance detection (UV detection) and ultraviolet-visible absorbance detection (UV-Vis detection); fluorescence detection (RF detection); differential refractive index detection (RI detection); electrical conductivity detection (CD detection); electrochemical detection (EC detection); mass spectrometry; and other known detection methods. Of these, absorbance detection is preferred because it can detect a wide range of light-absorbing substances such as aromatic compounds. Mass spectrometry (MS) is preferred because it allows various analyses such as qualitative analysis, quantitative analysis, structural analysis, and molecular weight measurement. It is also preferable to use absorbance detection and mass spectrometry in combination.

[0057] Here, it is known that, as a conventional technique, phosphate compounds and polycarboxylic acid compounds can be analyzed by liquid chromatography-mass spectrometry (LC-MS) using a mobile phase containing medronic acid (Anal. Chem. 2018, 90, 15, 9457-9464). However, because medronic acid is nonvolatile, using a mobile phase containing medronic acid poses the problem of detector contamination. In contrast, in the analytical method according to the present embodiment, the oxalic acid compounds and monocarboxylic acid compounds contained in the mobile phase are both volatile, which more effectively prevents contamination of the mass spectrometer (MS detector). Therefore, the analytical method according to the present embodiment can employ various detection methods, such as UV detection and MS detection, in the detection step.

[0058] The ionization method in mass spectrometry can be appropriately selected from electrospray ionization (ESI), atmospheric pressure chemical ionization (APCI), atmospheric pressure photoionization (APPI), fast atom bombardment (FAB), thermospray ionization (TSP), and other known ionization methods depending on the type of substance to be analyzed, etc.

[0059] The separation step and the detection step may be performed using a liquid chromatograph and a detection device in combination, or may be performed using a single separation and detection device, but are preferably performed using a single separation and detection device. This allows the analyte to be analyzed easily in a short time. Preferred examples of methods using a single separation and detection device include liquid chromatography-mass spectrometry (LC-MS) and liquid chromatography-tandem mass spectrometry (LC-MS / MS).

[0060] 1-6. Evaluation method

[0061] (Symmetry coefficient) Symmetry coefficient (P Ｓ ) is a coefficient indicating the degree of symmetry of the peak and is expressed by the following formula:

[0062]

[0063] W ０．０５ｈ denotes the peak width at 1 / 20 of the peak height from the baseline of the peak; ０．０５ｈ The distance from the peak apex to the horizontal axis, divided into two parts by a perpendicular line, is shown (see FIG. 1(A)).

[0064] Symmetry coefficient P Ｓ When P > 1, the peak is a tailing peak, and P Ｓ If P<1, the peak is determined to be a leading peak. Ｓ If ρ = 1, the peak is determined to be a symmetric Gaussian peak. The peak symmetry is determined by the symmetry coefficient P Ｓ The closer to 1, the higher the value.

[0065] (Number of theoretical plates) The number of theoretical plates (N) is an index that indicates column efficiency. The number of theoretical plates represents the number of plates in plate theory, and generally, the larger the number, the sharper the peak and the more efficient the column is. In a Gaussian distribution (normal distribution), the number of theoretical plates is expressed by the following formula:

[0066]

[0067] t Ｒ indicates retention time; W ０．５ｈindicates the peak width at half the height of the peak from the baseline of the peak, i.e., the peak half width (see FIG. 1(B)).

[0068] (Peak Half Width) The peak half width is one of the indicators of column efficiency. The peak half width is the peak width (W ０．５ｈ ) (see FIG. 1(B)), and the smaller this value, the better the column efficiency is judged to be.

[0069] (S / N ratio (signal / noise ratio)) The S / N ratio is the ratio of signal (S) to noise (N). It represents the ratio of the intensity (S) from the middle to the peak of the noise band to the width of the baseline (N). The higher the S / N ratio, the greater the signal intensity relative to the baseline, making it easier to detect peaks.

[0070] (Peak Area) The peak area refers to the area value of the detected peak. As described above, compounds having a phosphate group or polycarboxylic acid compounds have the problem of being adsorbed to metal piping and the like of a chromatograph. When these compounds are adsorbed to metal piping and the like, problems such as the peak area in the detector being smaller than the actual value or the peak area becoming unstable may occur. Therefore, the smaller the peak area, the more effectively the adsorption of these compounds to piping and the like can be suppressed. Furthermore, when measurements are performed multiple times, the smaller not only the peak area but also the standard deviation of the peak area, the more effectively the adsorption of these compounds to piping and the like can be suppressed.

[0071] 2. Mobile Phase Composition A second embodiment of the present disclosure is a composition used in a mobile phase for liquid chromatography, comprising one or more oxalic acid compounds selected from the group consisting of oxalic acid, oxalates represented by the following general formula (1), and oxalates represented by the following general formula (2), and one or more monocarboxylic acid compounds selected from the group consisting of formic acid, acetic acid, propionic acid, formates represented by the following general formula (3), acetates represented by the following general formula (4), and propionates represented by the following general formula (5). When the oxalic acid compound consists solely of oxalic acid, the monocarboxylic acid compound comprises one or more compounds selected from the group consisting of the formates, acetates, and propionates. When the monocarboxylic acid compound consists solely of one or more compounds selected from the group consisting of formic acid, acetic acid, and propionic acid, the oxalic acid compound comprises the oxalate. That is, in this embodiment, a salt is contained in either or both of the oxalic acid compound and the monocarboxylic acid compound.

[0072] (COONR ４ )2 (1) HOOCCOONR ４ (2) HCOONR ４ (3) CH ３ COONR ４ (4) CH ３ CH ２ COONR ４ (5) In the general formulas (1) to (5), each R independently represents a hydrogen atom or an alkyl group having 1 to 20 carbon atoms.

[0073] The mobile phase composition according to this embodiment can be used to prepare a mobile phase used in liquid chromatography in the analysis method according to the first embodiment. For example, the mobile phase can be prepared by dissolving the mobile phase composition according to this embodiment in a solvent for the mobile phase. The mobile phase composition may contain a portion of the solvent contained in the mobile phase according to the first embodiment, and may also contain any optional components that can be contained in the mobile phase.

[0074] Details of the oxalic acid compound, monocarboxylic acid compound, solvent, and optional components in this embodiment are as described in the description of the first embodiment. Therefore, general formulas (1) to (5) in this embodiment are synonymous with general formulas (1) to (5) in the first embodiment of the present disclosure. However, in this embodiment, the oxalic acid compound and the monocarboxylic acid compound may be hydrates.

[0075] 3. Analytical Kit A third embodiment of the present disclosure is an analytical kit including the mobile phase composition according to the second embodiment.

[0076] An example of an analytical kit according to this embodiment is shown in Fig. 2. In Fig. 2, analytical kit 10 includes mobile phase composition 11 stored in vial 13, but the type, size, and shape of the container storing mobile phase composition 11 are not particularly limited.

[0077] In addition to the mobile phase composition, the analysis kit 10 may also include any consumables used in liquid chromatography and detection.

[0078] The present disclosure will be explained in more detail below using examples, but the present disclosure is not limited to the following examples as long as it does not deviate from the gist of the disclosure.

[0079] Example 1 Preparation of Mobile Phase A 0.0905 g of oxalic acid and 0.3149 g of ammonium formate were dissolved in 500 mL of water. The pH (25°C) of the resulting aqueous solution was 4.0. This aqueous solution was mixed with acetonitrile in a ratio of 60:40 to obtain a mixed solution. The mixed solution was then degassed by irradiating it with ultrasound using a tabletop ultrasonic cleaner, to obtain mobile phase A. It was visually confirmed that oxalic acid and ammonium formate were completely dissolved in the resulting mobile phase A.

[0080] (Separation and Detection) A separation column packed with a stationary phase consisting of silica gel supported with a phosphobetaine polymer was connected to a liquid chromatograph. A sample containing 20 ppm of adenosine triphosphate (ATP) sodium salt was passed through the separation column together with mobile phase A, followed by detection using a UV detector and an MS detector. The liquid chromatography and detection conditions are shown below. This analysis and the same analysis were performed four more times. A representative chromatogram is shown in Figure 3. In addition, the theoretical plate number, peak half-width, symmetry factor, S / N ratio, and average and standard deviation of peak area values ​​were determined from five chromatograms using the methods described above. The results are shown in Table 1.

[0081] (Liquid Chromatography Conditions) Apparatus: Nexera series (Shimadzu Corporation) Separation column: DCpak PMPC (Daicel Corporation; 3 μm, PEEK liner column tube packed, 0.21 cmφ×15 cm) Injection volume: 1.0 μL Flow rate: 0.21 mL / min Column temperature: 40° C.

[0082] (UV detection conditions) UV detector: SPD-M20A (Shimadzu Corporation) Detection wavelength: 260 nm

[0083] (Mass spectrometry conditions) MS detector: LCMS-2020 (Shimadzu Corporation) Ionization method: Electrospray method Ionization mode: Positive mode Measurement mode: SIM (m / z = 507.90) Desolvation tube temperature: 250°C Heat block temperature: 400°C Dry gas flow rate: 15.0 mL / min Nebulizer flow rate: 1.5 mL / min

[0084] Comparative Example 1 (Preparation of Mobile Phase B) 0.0902 g of oxalic acid was dissolved in 500 mL of water. The pH (25°C) of the resulting aqueous solution was 2.7. A 28% aqueous ammonia solution was added to the aqueous solution to adjust the pH (25°C) of the aqueous solution to 4.0. This aqueous solution and acetonitrile were mixed in a 90:10 ratio to obtain a mixed solution. The mixed solution was then degassed by irradiating it with ultrasound using a tabletop ultrasonic cleaner to obtain mobile phase B. Complete dissolution of oxalic acid in the resulting mobile phase B was confirmed by visual observation. The composition of mobile phase B was adjusted so that the retention time of ATP sodium salt in liquid chromatography was similar to that of Example 1.

[0085] (Separation and Detection) Sample separation and detection were carried out five times in the same manner as in Example 1, except that mobile phase B was used instead of mobile phase A. A representative chromatogram is shown in Figure 4. In addition, the theoretical plate number, peak half-width, symmetry factor, S / N ratio, and average value and standard deviation of peak area values ​​were determined from the five chromatograms using the methods described above. The results are shown in Table 1.

[0086] Comparative Example 2 (Preparation of Mobile Phase C) 0.3150 g of ammonium formate was dissolved in 500 mL of water. The pH (25°C) of the resulting aqueous solution was 6.1. 98% formic acid was added to this aqueous solution to adjust the pH (25°C) of the aqueous solution to 4.0. This aqueous solution and acetonitrile were mixed in a ratio of 60:40. The mixture was then degassed by irradiating it with ultrasound using a tabletop ultrasonic cleaner to obtain mobile phase C. Complete dissolution of ammonium formate in the resulting mobile phase C was confirmed by visual observation. The composition of mobile phase C was adjusted so that the retention time of ATP sodium salt in liquid chromatography was similar to that of Example 1.

[0087] (Separation and Detection) Sample separation and detection were carried out five times in the same manner as in Example 1, except that mobile phase C was used instead of mobile phase A. A representative chromatogram is shown in Figure 5. In addition, the theoretical plate number, peak half-width, symmetry factor, S / N ratio, and average value and standard deviation of peak area values ​​were determined from the five chromatograms using the methods described above. The results are shown in Table 1.

[0088]

[0089] 3 to 5, in liquid chromatography, Example 1, which used a mobile phase prepared by dissolving oxalic acid and ammonium formate in water and acetonitrile, had a higher number of theoretical plates and a smaller peak half-width than Comparative Example 1, which used an oxalic acid solution as the mobile phase, and Comparative Example 2, which used an ammonium formate solution as the mobile phase, and therefore showed better column efficiency. Furthermore, Example 1 had a smaller symmetry factor than Comparative Examples 1 and 2, indicating that the symmetry of the peak shape of ATP sodium salt was excellent. Furthermore, in Example 1, the peak of ATP sodium salt was sharper than in Comparative Examples 2 and 3, and it was confirmed that the detection sensitivity was high in both UV detection and MS detection.

[0090] Furthermore, as shown in Table 1, Comparative Example 2, in which an ammonium formate solution was used as the mobile phase, had a large S / N ratio in the liquid chromatogram, and Comparative Example 1, in which an oxalic acid solution was used as the mobile phase, had the second largest S / N ratio after Comparative Example 2. On the other hand, Example 1, in which a solution prepared by dissolving oxalic acid and ammonium formate was used as the mobile phase, had a smaller S / N ratio than Comparative Examples 1 and 2, indicating that the signal intensity relative to the noise width was large.

[0091] In Example 1 and Comparative Example 1, the peak area values ​​were equivalent in both UV detection and MS detection, but Example 1 had a larger number of theoretical plates and S / N ratio, a smaller peak half-width, and a symmetry coefficient closer to 1 than Comparative Example 1. From these facts, it can be seen that in Example 1, high peak symmetry, column efficiency, detection sensitivity, and peak symmetry can be achieved by performing liquid chromatography using a mobile phase prepared by dissolving oxalic acid and ammonium formate.

[0092] Example 2 (Preparation of Mobile Phase D1) 0.0225 g of oxalic acid and 0.3153 g of ammonium formate were dissolved in 500 mL of water. The pH (25°C) of the resulting aqueous solution was 4.68. The aqueous solution was then degassed by irradiating it with ultrasonic waves using a tabletop ultrasonic cleaner to obtain Mobile Phase D1. It was visually confirmed that the oxalic acid and ammonium formate were completely dissolved in the resulting Mobile Phase D1.

[0093] (Preparation of Mobile Phase D2) Mobile phase D1 and acetonitrile were mixed in a ratio of 70:30 to obtain a mixed solution. The mixed solution was then degassed by irradiating it with ultrasound using a tabletop ultrasonic cleaner to obtain mobile phase D2. It was visually confirmed that oxalic acid and ammonium formate were completely dissolved in the obtained mobile phase D2.

[0094] (Separation and Detection) A separation column packed with a stationary phase consisting of silica gel supported with a phosphobetaine polymer was connected to a liquid chromatograph. A sample containing 100 ppm each of adenylic acid (AMP) sodium salt, adenosine diphosphate (ADP) sodium salt, and adenosine triphosphate (ATP) sodium salt was passed through the separation column together with mobile phases D1 and D2, followed by detection using a UV detector and an MS detector. AMP sodium salt, ADP sodium salt, and ATP sodium salt eluted in this order. The liquid chromatography and detection conditions are shown below. The chromatogram is shown in Figure 6. The theoretical plate number, peak half-width, symmetry factor, S / N ratio, and peak area value determined by the above-mentioned method are also shown in Table 2.

[0095] (Liquid Chromatography Conditions) Apparatus: Nexera series (Shimadzu Corporation) Separation column: DCpak PMPC (Daicel Corporation; 3 μm, PEEK liner column tube packed, 0.21 cmφ×15 cm) Injection volume: 1.0 μL Flow rate: 0.21 mL / min Column temperature: 40° C.

[0096] (Flow conditions for mobile phase and aqueous solution) 0 to 2 min: Mobile phase D1 / Mobile phase D2 = 0 / 100 2 to 20 min: Mobile phase D1 / Mobile phase D2 = 0 / 100 → 80 / 20 20 to 24 min: Mobile phase D1 / Mobile phase D2 = 80 / 20 24 to 25 min: Mobile phase D1 / Mobile phase D2 = 80 / 20 → 0 / 100 25 to 35 min: Mobile phase D1 / Mobile phase D2 = 0 / 100

[0097] (UV detection conditions) UV detector: SPD-M20A (Shimadzu Corporation) Detection wavelength: 260 nm

[0098] (Mass spectrometry conditions) MS detector: LCMS-2020 (Shimadzu Corporation) Ionization method: Electrospray method Ionization mode: Positive mode Measurement mode: SIM (Adenosine phosphates: Positive 348.05 m / z, 428.00 m / z, 508.00 m / z) Desolvation tube temperature: 250°C Heat block temperature: 400°C Dry gas flow rate: 15.0 mL / min Nebulizer flow rate: 1.5 mL / min

[0099] Example 3 Preparation of Mobile Phase E1 0.0450 g of oxalic acid and 0.3153 g of ammonium formate were dissolved in 500 mL of water. The pH (25°C) of the resulting aqueous solution was 4.38. The aqueous solution was then degassed by irradiating it with ultrasonic waves using a tabletop ultrasonic cleaner to obtain mobile phase E1. It was visually confirmed that oxalic acid and ammonium formate were completely dissolved in mobile phase E1.

[0100] (Preparation of Mobile Phase E2) Mobile phase E1 and acetonitrile were mixed in a ratio of 70:30 to obtain a mixed solution. The mixed solution was then degassed by irradiating it with ultrasound using a tabletop ultrasonic cleaner to obtain mobile phase E2. It was visually confirmed that oxalic acid and ammonium formate were completely dissolved in the obtained mobile phase E2.

[0101] (Separation and Detection) Sample separation and detection were carried out in the same manner as in Example 2, except that mobile phase E1 was used instead of mobile phase D1, and mobile phase E2 was used instead of mobile phase D2. A representative chromatogram is shown in Figure 7. The theoretical plate number, peak half-width, symmetry factor, S / N ratio, and peak area value obtained by the above-mentioned methods are shown in Table 2.

[0102] Example 4 Preparation of Mobile Phase F1 0.00450 g of oxalic acid and 0.3153 g of ammonium formate were dissolved in 500 mL of water. The pH (25°C) of the resulting aqueous solution was 5.24. The aqueous solution was then degassed by irradiating it with ultrasonic waves using a tabletop ultrasonic cleaner to obtain Mobile Phase F1. It was visually confirmed that the oxalic acid and ammonium formate were completely dissolved in the resulting Mobile Phase F1.

[0103] (Preparation of Mobile Phase F2) Mobile phase F1 and acetonitrile were mixed in a ratio of 70:30 to obtain a mixed solution. The mixed solution was then degassed by irradiating it with ultrasound using a tabletop ultrasonic cleaner to obtain mobile phase F2. It was visually confirmed that oxalic acid and ammonium formate were completely dissolved in the obtained mobile phase F2.

[0104] (Separation and detection) Sample separation and detection were carried out in the same manner as in Example 2, except that mobile phase F1 was used instead of mobile phase D1, and mobile phase F2 was used instead of mobile phase D2. A typical chromatogram is shown in Figure 8. The theoretical plate number, peak half-width, symmetry factor, S / N ratio, and peak area value obtained by the above-mentioned methods are shown in Table 2.

[0105] Example 5 Preparation of Mobile Phase G1 0.0225 g of oxalic acid and 0.6306 g of ammonium formate were dissolved in 500 mL of water. The pH (25°C) of the resulting aqueous solution was 4.96. The aqueous solution was then degassed by irradiating it with ultrasonic waves using a tabletop ultrasonic cleaner to obtain mobile phase G1. It was visually confirmed that oxalic acid and ammonium formate were completely dissolved in the resulting mobile phase G1.

[0106] (Preparation of Mobile Phase G2) Mobile phase G1 and acetonitrile were mixed in a ratio of 70:30 to obtain a mixed solution. The mixed solution was then degassed by irradiating it with ultrasound using a tabletop ultrasonic cleaner to obtain mobile phase G2. It was visually confirmed that oxalic acid and ammonium formate were completely dissolved in the resulting mobile phase G2.

[0107] (Separation and detection) Sample separation and detection were carried out in the same manner as in Example 2, except that mobile phase G1 was used instead of mobile phase D1, and mobile phase G2 was used instead of mobile phase D2. A typical chromatogram is shown in Figure 9. The theoretical plate number, peak half-width, symmetry factor, S / N ratio, and peak area value obtained by the above-mentioned methods are shown in Table 2.

[0108] Example 6 Preparation of Mobile Phase H1 0.0450 g of oxalic acid and 0.6306 g of ammonium formate were dissolved in 500 mL of water. The pH (25°C) of the resulting aqueous solution was 4.67. The aqueous solution was then degassed by irradiating it with ultrasonic waves using a tabletop ultrasonic cleaner to obtain mobile phase H1. It was visually confirmed that oxalic acid and ammonium formate were completely dissolved in the resulting mobile phase H1.

[0109] (Preparation of Mobile Phase H2) Mobile phase H1 and acetonitrile were mixed in a ratio of 70:30 to obtain a mixed solution. The mixed solution was then degassed by irradiating it with ultrasound using a tabletop ultrasonic cleaner to obtain mobile phase H2. It was visually confirmed that oxalic acid and ammonium formate were completely dissolved in the obtained mobile phase H2.

[0110] (Separation and detection) Sample separation and detection were carried out in the same manner as in Example 2, except that mobile phase H1 was used instead of mobile phase D1, and mobile phase H2 was used instead of mobile phase D2. A representative chromatogram is shown in Figure 10. The theoretical plate number, peak half-width, symmetry factor, S / N ratio, and peak area value obtained by the above-mentioned methods are also shown in Table 2.

[0111] Example 7 Preparation of Mobile Phase I1 0.00450 g of oxalic acid and 0.6306 g of ammonium formate were dissolved in 500 mL of water. The pH (25°C) of the resulting aqueous solution was 5.31. The aqueous solution was then degassed by irradiating it with ultrasound using a tabletop ultrasonic cleaner to obtain mobile phase I1. It was visually confirmed that oxalic acid and ammonium formate were completely dissolved in the resulting mobile phase I1.

[0112] (Preparation of Mobile Phase I2) Mobile phase I1 and acetonitrile were mixed in a ratio of 70:30 to obtain a mixed solution. The mixed solution was then degassed by irradiating it with ultrasound using a tabletop ultrasonic cleaner to obtain mobile phase I2. It was visually confirmed that oxalic acid and ammonium formate were completely dissolved in the obtained mobile phase I2.

[0113] (Separation and Detection) Sample separation and detection were carried out in the same manner as in Example 2, except that mobile phase I1 was used instead of mobile phase D1, and mobile phase I2 was used instead of mobile phase D2. A representative chromatogram is shown in Figure 11. The theoretical plate number, peak half-width, symmetry factor, S / N ratio, and peak area value obtained by the above-mentioned methods are shown in Table 2.

[0114] Example 8 Preparation of Mobile Phase J1 0.00225 g of oxalic acid and 0.1576 g of ammonium formate were dissolved in 500 mL of water. The pH (25°C) of the resulting aqueous solution was 4.44. The aqueous solution was then degassed by irradiating it with ultrasound using a tabletop ultrasonic cleaner to obtain mobile phase J1. It was visually confirmed that oxalic acid and ammonium formate were completely dissolved in the resulting mobile phase J1.

[0115] (Preparation of Mobile Phase J2) 0.00225 g of oxalic acid and 0.1576 g of ammonium formate were dissolved in 500 mL of water. The pH (25°C) of the resulting aqueous solution was 4.44. This aqueous solution was mixed with acetonitrile in a ratio of 70:30 to obtain a mixed solution. The mixed solution was then degassed by irradiating it with ultrasound using a tabletop ultrasonic cleaner, obtaining mobile phase J2. It was visually confirmed that oxalic acid and ammonium formate were completely dissolved in the resulting mobile phase J2.

[0116] (Separation and Detection) Sample separation and detection were carried out in the same manner as in Example 2, except that mobile phase J1 was used instead of mobile phase D1, and mobile phase J2 was used instead of mobile phase D2. A representative chromatogram is shown in Figure 12. The theoretical plate number, peak half-width, symmetry factor, S / N ratio, and peak area value obtained by the above-mentioned methods are shown in Table 2.

[0117] Example 9 (Preparation of Mobile Phase K1) 0.0450 g of oxalic acid and 0.1576 g of ammonium formate were dissolved in 500 mL of water. The pH (25°C) of the resulting aqueous solution was 4.08. The aqueous solution was then degassed by irradiating it with ultrasound using a tabletop ultrasonic cleaner to obtain mobile phase K1. It was visually confirmed that oxalic acid and ammonium formate were completely dissolved in the resulting mobile phase K1.

[0118] (Preparation of Mobile Phase K2) 0.0450 g of oxalic acid and 0.1576 g of ammonium formate were dissolved in 500 mL of water. The pH (25°C) of the resulting aqueous solution was 4.08. This aqueous solution was mixed with acetonitrile in a ratio of 70:30 to obtain a mixed solution. The mixed solution was then degassed by irradiating it with ultrasound using a tabletop ultrasonic cleaner, obtaining mobile phase K2. It was visually confirmed that oxalic acid and ammonium formate were completely dissolved in the resulting mobile phase K2.

[0119] (Separation and Detection) Sample separation and detection were carried out in the same manner as in Example 2, except that mobile phase K1 was used instead of mobile phase D1, and mobile phase K2 was used instead of mobile phase D2. A representative chromatogram is shown in Figure 13. The theoretical plate number, peak half-width, symmetry factor, S / N ratio, and peak area value obtained by the above-mentioned methods are shown in Table 2.

[0120] Example 10 (Preparation of Mobile Phase L1) 0.0045 g of oxalic acid and 0.1576 g of ammonium formate were dissolved in 500 mL of water. The pH (25°C) of the resulting aqueous solution was 5.22. The aqueous solution was then degassed by irradiating it with ultrasonic waves using a tabletop ultrasonic cleaner to obtain mobile phase L1. It was visually confirmed that oxalic acid and ammonium formate were completely dissolved in the resulting mobile phase L1.

[0121] (Preparation of mobile phase L2) Mobile phase L1 and acetonitrile were mixed in a ratio of 70:30 to obtain a mixed solution. The mixed solution was then degassed by irradiating it with ultrasound using a tabletop ultrasonic cleaner to obtain mobile phase L2. It was visually confirmed that oxalic acid and ammonium formate were completely dissolved in the obtained mobile phase L2.

[0122] (Separation and detection) Sample separation and detection were carried out in the same manner as in Example 2, except that mobile phase L1 was used instead of mobile phase D1, and mobile phase L2 was used instead of mobile phase D2. A representative chromatogram is shown in Figure 14. The theoretical plate number, peak half-width, symmetry factor, S / N ratio, and peak area value obtained by the above-mentioned methods are shown in Table 2.

[0123]

[0124] Example 11 (Preparation of Mobile Phase M1) 0.0225 g of oxalic acid and 0.3153 g of ammonium formate were dissolved in 500 mL of water. The pH (25°C) of the resulting aqueous solution was 4.68. The aqueous solution was then degassed by irradiating it with ultrasonic waves using a tabletop ultrasonic cleaner to obtain mobile phase M1. It was visually confirmed that oxalic acid and ammonium formate were completely dissolved in the resulting mobile phase M1.

[0125] (Preparation of Mobile Phase M2) Mobile phase M1 and acetonitrile were mixed in a ratio of 70:30 to obtain a mixed solution. The mixed solution was then degassed by irradiating it with ultrasound using a tabletop ultrasonic cleaner to obtain mobile phase M2. It was visually confirmed that oxalic acid and ammonium formate were completely dissolved in the obtained mobile phase M2.

[0126] (Separation and Detection) A separation column packed with a stationary phase consisting of silica gel supported with a phosphobetaine polymer was connected to a liquid chromatograph. A sample containing 100 ppm each of cytidylic acid (CMP) sodium salt, cytidine diphosphate (CDP) sodium salt, and cytidine triphosphate (CTP) sodium salt was passed through the separation column together with mobile phases M1 and M2, followed by detection using a UV detector and an MS detector. CMP sodium salt, CDP sodium salt, and CTP sodium salt eluted in this order. The liquid chromatography and detection conditions are shown below. The chromatogram is shown in Figure 15. The theoretical plate number, peak half-width, symmetry factor, S / N ratio, and peak area value determined by the above-mentioned methods are also shown in Table 3.

[0127] (Liquid Chromatography Conditions) Apparatus: Nexera series (Shimadzu Corporation) Separation column: DCpak PMPC (Daicel Corporation; 3 μm, PEEK liner column tube packed, 0.21 cmφ×15 cm) Injection volume: 1.0 μL Flow rate: 0.21 mL / min Column temperature: 40° C.

[0128] (Flow conditions for mobile phase and aqueous solution) 0 to 2 min: Mobile phase M1 / Mobile phase M2 = 0 / 100 2 to 20 min: Mobile phase M1 / Mobile phase M2 = 0 / 100 → 80 / 20 20 to 24 min: Mobile phase M1 / Mobile phase M2 = 80 / 20 24-25 min: Mobile phase M1 / Mobile phase M2 = 80 / 20 → 0 / 100 25-35 min: Mobile phase M1 / Mobile phase M2 = 0 / 100

[0129] (UV detection conditions) UV detector: SPD-M20A (Shimadzu Corporation) Detection wavelength: 272 nm

[0130] (Mass spectrometry conditions) MS detector: LCMS-2020 (Shimadzu Corporation) Ionization method: Electrospray method Ionization mode: Positive mode Measurement mode: SIM (Cytidine phosphates: Positive 324.00 m / z, 404.00 m / z, 483.95 m / z) Desolvation tube temperature: 250°C Heat block temperature: 400°C Dry gas flow rate: 15.0 mL / min Nebulizer flow rate: 1.5 mL / min

[0131] Example 12 (Preparation of Mobile Phase N1) 0.0450 g of oxalic acid and 0.3153 g of ammonium formate were dissolved in 500 mL of water. The pH (25°C) of the resulting aqueous solution was 4.38. The aqueous solution was then degassed by irradiating it with ultrasound using a tabletop ultrasonic cleaner to obtain Mobile Phase N1. It was visually confirmed that the oxalic acid and ammonium formate were completely dissolved in the resulting Mobile Phase N1.

[0132] (Preparation of Mobile Phase N2) Mobile phase N1 and acetonitrile were mixed in a ratio of 70:30 to obtain a mixed solution. The mixed solution was then degassed by irradiating it with ultrasound using a tabletop ultrasonic cleaner to obtain mobile phase N2. It was visually confirmed that oxalic acid and ammonium formate were completely dissolved in the obtained mobile phase N2.

[0133] (Separation and detection) Sample separation and detection were carried out in the same manner as in Example 11, except that mobile phase N1 was used instead of mobile phase M1, and mobile phase N2 was used instead of mobile phase M2. A representative chromatogram is shown in Figure 16. The theoretical plate number, peak half-width, symmetry factor, S / N ratio, and peak area value obtained by the above-mentioned methods are shown in Table 3.

[0134] Example 13 (Preparation of Mobile Phase O1) 0.00450 g of oxalic acid and 0.3153 g of ammonium formate were dissolved in 500 mL of water. The pH (25°C) of the resulting aqueous solution was 5.24. The aqueous solution was then degassed by irradiating it with ultrasound using a tabletop ultrasonic cleaner to obtain mobile phase O1. It was visually confirmed that oxalic acid and ammonium formate were completely dissolved in the resulting mobile phase O1.

[0135] (Preparation of Mobile Phase O2) Mobile phase O1 and acetonitrile were mixed in a ratio of 70:30 to obtain a mixed solution. The mixed solution was then degassed by irradiating it with ultrasound using a tabletop ultrasonic cleaner to obtain mobile phase O2. It was visually confirmed that oxalic acid and ammonium formate were completely dissolved in the obtained mobile phase O2.

[0136] (Separation and detection) Sample separation and detection were carried out in the same manner as in Example 11, except that mobile phase O1 was used instead of mobile phase M1, and mobile phase O2 was used instead of mobile phase M2. A representative chromatogram is shown in Figure 17. The theoretical plate number, peak half-width, symmetry factor, S / N ratio, and peak area value obtained by the above-mentioned methods are shown in Table 3.

[0137] Example 14 (Preparation of Mobile Phase P1) 0.0225 g of oxalic acid and 0.6306 g of ammonium formate were dissolved in 500 mL of water. The pH (25°C) of the resulting aqueous solution was 4.96. The aqueous solution was then degassed by irradiating it with ultrasound using a tabletop ultrasonic cleaner to obtain mobile phase P1. It was visually confirmed that oxalic acid and ammonium formate were completely dissolved in the resulting mobile phase P1.

[0138] (Preparation of mobile phase P2) Mobile phase P1 and acetonitrile were mixed in a ratio of 70:30 to obtain a mixed solution. The mixed solution was then degassed by irradiating it with ultrasound using a tabletop ultrasonic cleaner to obtain mobile phase P2. It was visually confirmed that oxalic acid and ammonium formate were completely dissolved in the obtained mobile phase P2.

[0139] (Separation and detection) Sample separation and detection were carried out in the same manner as in Example 11, except that mobile phase P1 was used instead of mobile phase M1, and mobile phase P2 was used instead of mobile phase M2. A representative chromatogram is shown in Figure 18. The theoretical plate number, peak half-width, symmetry factor, S / N ratio, and peak area value obtained by the above-mentioned methods are shown in Table 3.

[0140] Example 15 (Preparation of Mobile Phase Q1) 0.0450 g of oxalic acid and 0.6306 g of ammonium formate were dissolved in 500 mL of water. The pH (25°C) of the resulting aqueous solution was 4.67. The aqueous solution was then degassed by irradiating it with ultrasonic waves using a tabletop ultrasonic cleaner to obtain mobile phase Q1. It was visually confirmed that oxalic acid and ammonium formate were completely dissolved in the resulting mobile phase Q1.

[0141] (Preparation of Mobile Phase Q2) Mobile phase Q1 and acetonitrile were mixed in a ratio of 70:30 to obtain a mixed solution. The mixed solution was then degassed by irradiating it with ultrasound using a tabletop ultrasonic cleaner to obtain mobile phase Q2. It was visually confirmed that oxalic acid and ammonium formate were completely dissolved in the resulting mobile phase Q2.

[0142] (Separation and detection) Sample separation and detection were carried out in the same manner as in Example 11, except that mobile phase Q1 was used instead of mobile phase M1, and mobile phase Q2 was used instead of mobile phase M2. A representative chromatogram is shown in Figure 19. The theoretical plate number, peak half-width, symmetry factor, S / N ratio, and peak area value obtained by the above-mentioned methods are shown in Table 3.

[0143] Example 16 (Preparation of Mobile Phase R1) 0.00450 g of oxalic acid and 0.6306 g of ammonium formate were dissolved in 500 mL of water. The pH (25°C) of the resulting aqueous solution was 5.31. The aqueous solution was then degassed by irradiating it with ultrasound using a tabletop ultrasonic cleaner to obtain mobile phase R1. It was visually confirmed that oxalic acid and ammonium formate were completely dissolved in the resulting mobile phase R1.

[0144] (Preparation of mobile phase R2) Mobile phase R1 and acetonitrile were mixed in a ratio of 70:30 to obtain a mixed solution. The mixed solution was then degassed by irradiating it with ultrasound using a tabletop ultrasonic cleaner to obtain mobile phase R2. It was visually confirmed that oxalic acid and ammonium formate were completely dissolved in the obtained mobile phase R2.

[0145] (Separation and detection) Sample separation and detection were carried out in the same manner as in Example 11, except that mobile phase R1 was used instead of mobile phase M1, and mobile phase R2 was used instead of mobile phase M2. A representative chromatogram is shown in Figure 20. The theoretical plate number, peak half-width, symmetry factor, S / N ratio, and peak area value obtained by the above-mentioned methods are shown in Table 3.

[0146] Example 17 (Preparation of Mobile Phase S1) 0.0225 g of oxalic acid and 0.1576 g of ammonium formate were dissolved in 500 mL of water. The pH (25°C) of the resulting aqueous solution was 4.44. The aqueous solution was then degassed by irradiating it with ultrasonic waves using a tabletop ultrasonic cleaner to obtain mobile phase S1. It was visually confirmed that oxalic acid and ammonium formate were completely dissolved in the resulting mobile phase S1.

[0147] (Preparation of mobile phase S2) Mobile phase S1 and acetonitrile were mixed in a ratio of 70:30 to obtain a mixed solution. The mixed solution was then degassed by irradiating it with ultrasound using a tabletop ultrasonic cleaner to obtain mobile phase S2. It was visually confirmed that oxalic acid and ammonium formate were completely dissolved in the obtained mobile phase S2.

[0148] (Separation and detection) Sample separation and detection were carried out in the same manner as in Example 11, except that mobile phase S1 was used instead of mobile phase M1, and mobile phase S2 was used instead of mobile phase M2. A representative chromatogram is shown in Figure 21. The theoretical plate number, peak half-width, symmetry factor, S / N ratio, and peak area value obtained by the above-mentioned methods are also shown in Table 3.

[0149] Example 18 (Preparation of Mobile Phase T1) 0.0450 g of oxalic acid and 0.1576 g of ammonium formate were dissolved in 500 mL of water. The pH (25°C) of the resulting aqueous solution was 4.09. The aqueous solution was then degassed by irradiating it with ultrasonic waves using a tabletop ultrasonic cleaner to obtain mobile phase T1. It was visually confirmed that oxalic acid and ammonium formate were completely dissolved in the resulting mobile phase T1.

[0150] (Preparation of mobile phase T2) Mobile phase T1 and acetonitrile were mixed in a ratio of 70:30 to obtain a mixed solution. The mixed solution was then degassed by irradiating it with ultrasonic waves using a tabletop ultrasonic cleaner to obtain mobile phase T2. It was visually confirmed that oxalic acid and ammonium formate were completely dissolved in the obtained mobile phase T2.

[0151] (Separation and detection) Sample separation and detection were carried out in the same manner as in Example 11, except that mobile phase T1 was used instead of mobile phase M1, and mobile phase T2 was used instead of mobile phase M2. A representative chromatogram is shown in Figure 22. The theoretical plate number, peak half-width, symmetry factor, S / N ratio, and peak area value obtained by the above-mentioned methods are shown in Table 3.

[0152] Example 19 (Preparation of Mobile Phase U1) 0.0045 g of oxalic acid and 0.1576 g of ammonium formate were dissolved in 500 mL of water. The pH (25°C) of the resulting aqueous solution was 5.22. The aqueous solution was then degassed by irradiating it with ultrasound using a tabletop ultrasonic cleaner to obtain mobile phase U1. It was visually confirmed that oxalic acid and ammonium formate were completely dissolved in the resulting mobile phase U1.

[0153] (Preparation of Mobile Phase U2) Mobile phase U1 and acetonitrile were mixed in a ratio of 70:30 to obtain a mixed solution. The mixed solution was then degassed by irradiating it with ultrasound using a desktop ultrasonic cleaner to obtain mobile phase U2. It was visually confirmed that oxalic acid and ammonium formate were completely dissolved in the resulting mobile phase U2.

[0154] (Separation and detection) Sample separation and detection were carried out in the same manner as in Example 11, except that mobile phase U1 was used instead of mobile phase M1, and mobile phase U2 was used instead of mobile phase M2. A representative chromatogram is shown in Figure 23. The theoretical plate number, peak half-width, symmetry factor, S / N ratio, and peak area value obtained by the above-mentioned methods are shown in Table 3.

[0155]

[0156] 10: Analysis kit 11: Mobile phase composition 13: Vial

Claims

1. A method for analyzing a sample, comprising: a separation step of separating an analyte contained in the sample by chromatography; and a detection step of detecting the analyte separated in the separation step, wherein the mobile phase used in the chromatography is composed of one or more oxalic acid compounds selected from the group consisting of oxalic acid, oxalates represented by the following general formula (1), and oxalates represented by the following general formula (2), and formic acid, acetic acid, propionic acid, formates represented by the following general formula (3), acetates represented by the following general formula (4), and propionates represented by the following general formula (5). When the oxalic acid compound consists only of oxalic acid, the monocarboxylic acid compound includes one or more selected from the group consisting of the formate, the acetate, and the propionate. When the monocarboxylic acid compound consists only of one or more selected from the group consisting of formic acid, acetic acid, and propionic acid, the oxalic acid compound includes the oxalate. (COONR ４ )2 (1) HOOCCCOONR ４ (2) HCOONR ４ (3) CH ３ COONR ４ (4) CH ３ CH ２ COONR ４ (5) (In the formula, each R independently represents a hydrogen atom or an alkyl group having 1 to 20 carbon atoms.) 2. The analysis method according to claim 1, wherein the sample contains a compound having a phosphate group, a mixture of enantiomers, or a polycarboxylic acid compound.

3. The analysis method according to claim 2, wherein the compound having a phosphate group is at least one selected from the group consisting of nucleic acids, nucleic acid derivatives, nucleotides, and nucleotide derivatives.

4. The analysis method according to claim 1, wherein the chromatography is liquid chromatography or supercritical fluid chromatography.

5. The analysis method according to claim 1, wherein the detection of the analyte in the detection step is performed by absorbance detection, mass spectrometry, or a combination thereof.

6. The analysis method according to claim 5, wherein the ionization method in the mass spectrometry is an electrospray method or an atmospheric pressure chemical ionization method.

7. The analysis method according to claim 1, wherein the mobile phase satisfies any one or both of the following (a) and (b): (a) The concentration of the oxalic acid compound is 0.01 mM or more and 1.0 mM or less. (b) The concentration of the monocarboxylic acid compound is 3.5 mM or more and 20.0 mM or less.

8. A composition used as a mobile phase in chromatography, comprising at least one oxalic acid compound selected from the group consisting of oxalic acid, an oxalate represented by the following general formula (1), and an oxalate represented by the following general formula (2), and at least one monocarboxylic acid compound selected from the group consisting of formic acid, acetic acid, propionic acid, a formate represented by the following general formula (3), an acetate represented by the following general formula (4), and a propionate represented by the following general formula (5). When the oxalic acid compound consists only of oxalic acid, the monocarboxylic acid compound includes at least one selected from the group consisting of the formate, the acetate, and the propionate. When the monocarboxylic acid compound consists only of at least one selected from the group consisting of formic acid, acetic acid, and propionic acid, the oxalic acid compound includes the oxalate. A composition for a mobile phase. (COONR ４ )2 (1) HOOCC OONR ４ (2) HCOONR ４ (3) CH ３ COONR ４ (4) CH ３ CH ２ COONR ４ (5) (In the formula, each R independently represents a hydrogen atom or an alkyl group having 1 to 20 carbon atoms.) 9. An analysis kit containing the composition for the mobile phase according to claim 8.

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